Validation of the Legionella pneumophila SG1 DETECT Kit for Quantification of Legionella pneumophila Serogroup 1 Bacteria in Potable Waters, Process Waters, and Surface Waters: AOAC Performance Tested MethodSM 052002.

The L.p.SG1 DETECT Kit is a rapid, quantitative method for the detection and enumeration of Legionella pneumophila serogroup 1 (L.p. SG1) bacteria from different water matrixes. The method is based on a combination of immunomagnetic separation (IMS) and flow cytometric (FCM) quantification. To this end, the method employs magnetic particles conjugated to anti-L.p. SG1 antibodies for the IMS of the target bacteria from environmental matrices and fluorescently labeled anti-L.p. SG1 antibodies for subsequent quantification by FCM. The IMS can be performed either manually with a magnetic rack (rqmicro.MIMS) or automated with the rqmicro.STREAM sample preparation instrument. Compared to the reference method ISO 11731:2017, which is based on culturing and enumeration of colony forming units (CFU) on agar plates, and can take up to 10 days until results are available, analysis with the L.p. SG1 DETECT Kit is culture-independent and delivers results within 2 h. This Performance Tested Method validation study demonstrates a robust method with recoveries exceeding 69%, inclusivity of 100%, exclusivity of 97.2%, and a shelf life of at least 6 months at 4°C or 40 days at 25°C. The Limit of Detection (LOD) was determined at 21 CFU/L and the Limit of Quantification (LOQ) at 80 CFU/L for potable water using the rqmicro.STREAM. The matrix study across three different types of water matrixes (potable, surface, and industrial process water), demonstrates superior repeatability and reproducibility, as well as equivalent or even superior detection of L.p. SG1 bacteria compared to the standard ISO 11731 method.

Interaction of the gp120 V1V2 loop with a neighboring gp120 unit shields the HIV envelope trimer against cross-neutralizing antibodies.

The HIV-1 envelope trimer adopts a quaternary conformation that effectively shields neutralization-sensitive domains and thus represents a major obstacle for natural and vaccine-elicited antibody responses. By using a structure-function analysis based on a specifically devised mathematical model, we demonstrate in this study that protection from neutralization is enforced by intersubunit contact between the variable loops 1 and 2 (V1V2) and domains of neighboring gp120 subunits in the trimer encompassing the V3 loop. Our data are consistent with an interaction of the V1V2 and V3 loop at the spike apex as proposed by cryoelectron tomography experiments. By defining the orientation of the V1V2 loop within the trimer toward the neighboring gp120 subunit's V3 loop, our data close an important gap in the understanding of the architecture of the trimeric spike. Knowledge on how the V1V2 barrier functions in the context of the trimer to mask conserved epitopes on gp120 may aid future vaccine design.

Th cells act via two synergistic pathways to promote antiviral CD8+ T cell responses.

The mechanisms of how Th cells promote CD8(+) T cell responses during viral infections are largely unknown. In this study, we unraveled the mechanisms of T cell help for CD8(+) T cell responses during vaccinia virus infection. Our results demonstrate that Th cells promote vaccinia virus-specific CD8(+) T cell responses via two interconnected synergistic pathways: First, CD40L expressed by activated CD4(+) T cells instructs dendritic cells to produce bioactive IL-12p70, which is directly sensed by Ag-specific CD8(+) T cells, resulting in increased IL-2Rα expression. Second, Th cells provide CD8(+) T cells with IL-2, thereby enhancing their survival. Thus, Th cells are at the center of an important communication loop with a central role for IL-2/IL-2R and bioactive IL-12.